Rapid and tunable post-translational coupling of genetic circuits

Protease competition is used to produce rapid and tunable coupling of genetic circuits, enabling a coupled clock network that can encode independent environmental cues into a single time series output, a form of frequency multiplexing in a genetic circuit context. Engineered biocircuits that can stand the noise A major challenge in synthetic biology is that of integrating individual regulatory modules into larger circuits in a noisy and highly interconnected cellular environment. Jeff Hasty and colleagues tackle this problem by recruiting a host-cell protein-degradation system to synchronize genetic circuits and obtain delay times much shorter than those achieved by transcription-based coupling. They then apply this post-translational engineering scheme to synchronize cellular clocks and enable bacterial colonies to respond to independent environmental cues in 'frequency multiplexing'. One promise of synthetic biology is the creation of genetic circuitry that enables the execution of logical programming in living cells. Such 'wet programming' is positioned to transform a wide and diverse swathe of biotechnology ranging from therapeutics and diagnostics to water treatment strategies. Although progress in the development of a library of genetic modules continues apace.sup.1,2,3,4, a major challenge for their integration into larger circuits is the generation of sufficiently fast and precise communication between modules.sup.5,6. An attractive approach is to integrate engineered circuits with host processes that facilitate robust cellular signalling.sup.7. In this context, recent studies have demonstrated that bacterial protein degradation can trigger a precise response to stress by overloading a limited supply of intracellular proteases.sup.8,9,10. Here we use protease competition to engineer rapid and tunable coupling of genetic circuits across multiple spatial and temporal scales. We characterize coupling delay times that are more than an order of magnitude faster than standard transcription-factor-based coupling methods (less than 1 min compared with ~20-40 min) and demonstrate tunability through manipulation of the linker between the protein and its degradation tag. We use this mechanism as a platform to couple genetic clocks at the intracellular and colony level, then synchronize the multi-colony dynamics to reduce variability in both clocks. We show how the coupled clock network can be used to encode independent environmental inputs into a single time series output, thus enabling frequency multiplexing (information transmitted on a common channel by distinct frequencies) in a genetic circuit context. Our results establish a general framework for the rapid and tunable coupling of genetic circuits through the use of native 'queueing' processes such as competitive protein degradation.
Author(s): Arthur Prindle [sup.1] , Jangir Selimkhanov [sup.1] , Howard Li [sup.1] , Ivan Razinkov [sup.1] , Lev S. Tsimring [sup.2] , Jeff Hasty [sup.1] [sup.2] [sup.3] Author Affiliations: (1) [...]